Novel E. coli 0157:H7 bacteriophage and uses thereof

ABSTRACT

The present invention is directed to isolated bacteriophage having strong lytic activity against strains of  E. coli  O157:H7, and methods of using that bacteriophage, and/or progeny or derivatives derived therefrom, to control the growth of  E. coli  O157:H7 in various settings.

FIELD OF THE INVENTION

The present invention relates to a novel bacteriophage designated ECML-4(the “Deposited bacteriophage”), and compositions and preparationscorresponding thereto. More specifically, isolated bacteriophagepreparations possessing lytic activity against strains of Escherichiacoli O157:H7 (the “Targeted Bacteria”) are provided in order to controlthe growth of the Targeted Bacteria, which will reduce their ability tocontaminate and colonize various environments, including but not limitedto (i) raw, unprocessed food products; (ii) equipment used to process ormanufacture various food products; (iii) various food products processedor manufactured with equipment contaminated with the Targeted Bacteria;(iv) animals contaminated with the Targeted Bacteria; and (v) animalenvironments contaminated with the Targeted Bacteria. The invention alsoprovides methods for detecting the presence of the Targeted Bacteria inprocessed or unprocessed food products, and in equipment used to processor manufacture the food products. In addition, the invention providesmethods of using the Deposited bacteriophage to remove the TargetedBacteria from medical, veterinary, animal husbandry, and otherenvironments where they may be passed to humans or animals. Also, theinvention additionally provides methods of using the bacteriophage toprevent and treat animal and human diseases caused by the TargetedBacteria.

BACKGROUND OF THE INVENTION

Bacteriophages are bacterial viruses that attach to their specific hostsand kill them by internal replication and bacterial lysis involving acomplex lytic cycle involving several structural and regulatory genes.Phages are very specific in that they only attack their targetedbacterial hosts. They cannot infect human or other eukaryotic cells.Bacteriophages were first identified, in the early part of the 20thcentury by Frederick Twort and Felix d'Herelle who called thembacteriophages or bacteria-eaters (from the Greek phago meaning to eator devour) (Duckworth, D. H. (1976). Who discovered bacteriophage?Bacteriol Rev 40(4): 793-802; Summers, W. C. (1999). Bacteriophagediscovered. Felix d'Herelle and the origins of molecular biology. NewHaven, Conn., Yale University Press: 47-59). At that time, with the ageof antibiotics still in the future, bacteriophages were considered to bea potentially powerful cure for bacterial infections, and they weretherapeutically utilized throughout the world during the pre-antibioticera. The use of phages in humans was found to be very safe; however,phage therapy did not always work and, with the advent of antibioticsthat were effective against a broad spectrum of pathogenic bacteria, itgradually fell out of favor in the United States and Western Europe.Several factors (reviewed in more detail in Sulakvelidze, A., Z.Alavidze, et al. (2001). Bacteriophage therapy. Antimicrob AgentsChemother 45(3): 649-659; Summers, W. C. (2001). Bacteriophage therapy.Ann Rev Microbiol 55: 437-51), including the lack of a broadunderstanding of phage biology and inadequate diagnostic bacteriologytechniques, contributed to the failure of some of the early phagetherapy studies and to the associated decline of interest in phagetherapy in the West. At the same time, phage therapy continued to beutilized in the former Soviet Union and Eastern Europe, where phagetherapy still is being used to treat a wide range of bacterial diseasesranging from intestinal infections to septicemia. Comprehensiveinformation about human and veterinary applications of bacteriophageshas been recently reviewed by several investigators (Alisky, J., K.Iczkowski, et al. (1998). Bacteriophages show promise as antimicrobialagents. J Infect 36(1): 5-15; Summers, W. C. (2001). Bacteriophagetherapy. Annu Rev Microbiol 55: 437-51; Merril, C. R., D. Scholl, et al.(2003). “The prospect for bacteriophage therapy in Western medicine.”Nat Rev Drug Discov 2(6): 489-497; Sulakvelidze, A. and P. Barrow(2005). Phage therapy in animals and agribusiness. Bacteriophages:Biology and Applications. E. Kutter and A. Sulakvelidze. Boca Raton,Fla., CRC Press: 335-380; Sulakvelidze, A. and E. Kutter (2005).Bacteriophage therapy in humans. Bacteriophages: Biology andApplication. E. Kutter and A. Sulakvelidze. Boca Raton, Fla., CRC Press:381-436).

Despite the use of bacteriophage in various practical settings,including the treatment of diseases in various animals, there remains inthe art a need for the discovery of novel bacteriophages, selection ofoptimal bacteriophages for specific practical applications, andidentifying methods for using these bacteriophages in several criticalareas, including clinical applications, food safety-related uses andenvironmental decontamination. For example, one significant needconcerns the treatment of processed or unprocessed food products toreduce, eliminate or prevent colonization with undesirable bacteria suchas pathogens responsible for food-borne illness and food spoilageorganisms. A second critical area of need concerns the removal ofundesirable bacteria from industrial environments such as foodprocessing facilities to prevent colonization thereof. A third criticalarea of need concerns the removal of antibiotic resistant organisms fromenvironments where they may be passed to susceptible humans and animals,such as hospitals, nursing homes, veterinary facilities, and other suchenvironments. Additionally, new bacteriophage and methods of using thesame are needed for the prevention or treatment of animal and humanbacterial disease, particularly those diseases caused byantibiotic-resistant organisms.

SUMMARY OF THE INVENTION

The invention meets the described needs and more by providingcompositions comprising novel ECML-4 bacteriophage having lyticspecificity for the Targeted Bacteria. The invention additionallyprovides methods of using the Deposited bacteriophage to control orprevent the infection or colonization of processed and unprocessed foodproducts by Targeted Bacteria, or colonization of equipment involved inthe processing of the same food product(s). The invention additionallyprovides methods of using the Deposited bacteriophage to prevent,eradicate, or reduce the levels of colonization of various animals(including humans) with Targeted Bacteria. The invention also providesmethods of detecting the presence of Targeted Bacteria cells onprocessed or unprocessed food products, or equipment involved in theprocessing of the same food products. The invention additionallyprovides methods of using the Deposited bacteriophage for the removal ofantibiotic-resistant or other undesirable pathogens from medical,veterinary, animal husbandry, and other environments where they may bepassed to humans or animals. The invention additionally provides formethods of using the Deposited bacteriophage to prevent or treat humanand/or other animal diseases caused by Targeted Bacteria

BRIEF DESCRIPTION OF THE FIGURES Figures

FIG. 1 shows a Restriction Fragment Length Polymorphism (RFLP) profileof the ECML-4 bacteriophage in comparison to two other bacteriophagesalso specific for the Targeted Bacteria.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

As used herein, “isolated” will mean material removed from its originalenvironment in which it naturally occurs, and thus is altered by thehand of man from its natural environment. Isolated material may be, forexample, foreign nucleic acid included in a vector system, foreignnucleic acid contained within a host cell, or any material which hasbeen removed from its original environment and thus altered by the handof man. Isolated material further encompasses bacteriophage specific forthe Targeted Bacteria or particular Targeted Bacteria isolates, isolatedand cultured separately from the environment in which it was located,where these isolates are present in purified compositions that do notcontain any significant amount of other bacteriophage or bacterialstrains, respectively.

As used therein, “Deposited bacteriophage” will mean isolatedbacteriophage ECML-4 deposited with the ATCC on Oct. 24, 2006, andreceiving ATCC Deposit Accession No. PTA-7948.

As used therein, “Targeted Bacteria” will mean E. coli O157:H7.

As used herein, “progeny” shall mean replicates of the Depositedbacteriophage, including descendants of the Deposited bacteriophagecreated by serial passage of the Deposited bacteriophage or by othermeans well known in the art, or bacteriophage whose RFLP profiles aresubstantially equivalent to the RFLP profile of the Depositedbacteriophage (See FIG. 1). The term substantially equivalent is used todescribe variability between organisms in accordance with the standardsadvanced by Tenover et al. from the United States Centers for DiseaseControl and Prevention (Tenover, F. C. et al. (1995) InterpretingChromosomal DNA Restriction Patterns Produced by Pulsed-Field GelElectrophoresis: Criteria for Bacterial Strain Typing. J. Clin.Microbiol. 33:2233-2239). Tenover et al. teaches the acceptable levelsof variation that may be seen when the genomes of identical propagatedorganisms are electrophoretically analyzed following restriction enzymedigestion.

As used herein, “recombinant bacteriophage” shall mean all geneticallymodified versions of the Deposited bacteriophage or its progeny,obtained by serial passaging (in vivo or in vitro) or geneticmanipulations of the Deposited bacteriophage or its progeny. Suchmanipulations include, but are not limited to, introducing genes or genecassettes encoding alternative proteins or nonfunctional proteins, ornoncoding nucleotide sequences into the genome of the Depositedbacteriophage.

As used herein, “derivatives” shall mean all substances that constitutesubunits or expression products of the Deposited bacteriophage or itsprogeny, including (but not limited to) phage nucleic acids, partial orcomplete phage genes, gene expression products, and structuralcomponents. For example, derivatives of the invention meanpolyribonucleotide(s) and polydeoxyribonucleotide(s), including modifiedor unmodified bacteriophage DNA, cDNA, mRNA and synthetic polynucleotidesequences, as well as DNA/RNA hybrids. Polynucleotides of the inventionalso encompass modified polynucleotides, such as for examplephosphorylated DNAs.

As used herein, “substantially pure” will mean material essentially freeof any similar macromolecules or other biological entities that wouldnormally be found with it in nature. For example, a substantially purebacteriophage is in a composition that contains no more than 1% of otherbacteriophages.

As used herein, “bacteriophage composition” will mean a compositioncomprising, or alternatively consisting essentially of, or alternativelyconsisting of, the Deposited bacteriophage. A “bacteriophagecomposition” as used herein does not include the Deposited bacteriophageas it exists in its natural environment prior to isolation and/orsubstantial purification.

As used herein, “colonization” or “colonized” will refer to the presenceof Targeted Bacteria on foodstuff(s), or environmental surface(s), or invivo such as in the gastrointestinal tract or skin of a mammalianorganism without perceptible significant alteration other than thepresence of bacteria. The terms “colonization” and “colonized” stand incontrast to the terms “infection” or “infected” which are commonlyunderstood to require perceptible deleterious alteration as part oftheir definition. “Colonization” and “colonized” may also refer to thepresence of bacteria in or on a human or animal without perceptibledamage, alteration, or disease.

As used herein, “ATCC” will mean the American Type Culture Collection,located at 10801 University Boulevard, Manassas, Va., 20110-2209, USA.

As used herein, “ORF” will mean an Open Reading Frame which is anin-frame sequence of codons that (in view of the genetic code)correspond to or encode a protein or peptide sequence. Two ORFscorrespond to each other if the sequences or their complementarysequences encode the same amino acid sequences. An ORF sequence,operably associated with appropriate regulatory sequences, may betranscribed and translated into a polypeptide in vivo. A polyadenylationsignal and transcription termination sequence will usually be located 3′to the coding sequence.

The Deposited Bacteriophage

The Deposited Bacteriophage has binding specificity for TargetedBacteria, and is capable of lysing Targeted Bacteria. The inventionfurther contemplates variants of the Deposited Bacteriophage, which arebacteriophage having minor variation(s) in the genomic sequence andpolypeptides encoded thereby while retaining the same general genotypicand phenotypic characteristics as the Deposited Bacteriophage. Suchvariants are considered to be the Deposited Bacteriophage in accordancewith the standards advanced by Tenover et al. from the United StatesCenters for Disease Control and Prevention (Tenover, F. C. et al. (1995)Interpreting Chromosomal DNA Restriction Patterns Produced byPulsed-Field Gel Electrophoresis: Criteria for Bacterial Strain Typing.J. Clin. Microbiol. 33:2233-2239). The invention also contemplatesprogeny and bacteriophage derivative(s).

The invention contemplates the use of the Deposited Bacteriophage, andits progeny and derivatives, to control the growth on, or colonizationof, processed and unprocessed food products by Targeted Bacteria, or thecolonization of buildings and equipment, particularly those associatedwith the processing of the same food product. The invention alsoprovides methods of identifying Targeted Bacteria as a bacterialdiagnostic and/or detecting the presence of Targeted Bacteria onprocessed or unprocessed food products, or equipment or buildings suchas those involved in the processing of the same food products. Theinvention further provides methods of using the Deposited Bacteriophagefor the removal of antibiotic-resistant or other undesirable pathogensfrom medical, veterinary, animal husbandry, or any additionalenvironments where they may be passed to humans or animals. Theinvention additionally provides for methods of using the DepositedBacteriophage to prevent and/or treat human and animal diseases causedby Targeted Bacteria. The Deposited Bacteriophage is administered forthe methods of the invention as a homogenous phage administration, oralternatively as a component of a multi-phage composition comprisingseveral bacteriophages. These methods of use are provided with greaterparticularity infra.

Use of the Deposited Bacteriophage and its Progeny Food Preservation

In one embodiment, the invention contemplates a method for theprevention of foodborne illnesses caused by the Targeted Bacteria,comprising contacting a food product or products with a microbial growthinhibiting effective amount of a bacteriophage composition comprisingthe Deposited Bacteriophage. The modes of contact include, but are notlimited to, spraying or misting the Deposited Bacteriophage compositionon the food product(s), or by dipping or soaking the food product(s) ina solution containing a concentration of the Deposited Bacteriophagesufficiently high to inhibit the growth of Targeted Bacteria, or adding,injecting or inserting the Deposited Bacteriophage into the foodproduct(s).

In another embodiment, the invention contemplates the application of theDeposited Bacteriophage composition to equipment associated with theprocessing of food product(s), such as cutting instruments, conveyorbelts, and any other implements utilized in the mass production of foodproducts, including the preparation, storage and packaging steps of foodprocessing. The Deposited Bacteriophage can additionally be introducedinto packaging materials used to contain food product(s), prior to orfollowing transfer of the food product(s) to the packaging materials.Alternatively the Deposited Bacteriophage can be useful in the localprocessing of food products located, for example, in the home or in arestaurant kitchen, using the same modes of contact as described supra.

In another embodiment of the invention, the Deposited Bacteriophage isadded as a component of paper products, either during processing orafter completion of processing of the paper products. Paper products towhich the Deposited Bacteriophage may be added include, but are notlimited to, paper towels, toilet paper, moist paper wipes. In apreferred embodiment of the invention, the Deposited Bacteriophage isadded as a component of cleansing wipes. The Deposited Bacteriophage maybe added in an aqueous state to a liquid-saturated paper product, oralternatively may be added in powder form, such as lyophilized, to drypaper products, or any combination thereof. In similar manner, theDeposited Bacteriophage may be incorporated into films such as thoseused for packaging foods, such as by impregnating or coating the film.

The methods of the invention further contemplate the application of theDeposited Bacteriophage to the floors, walls, ceilings, drains, or otherenvironmental surfaces in structures such as the industrial foodprocessing, military, or home environments. In a particularly preferredembodiment of the invention, the Deposited Bacteriophage is applied torefrigerated devices used to store or transport food or food products,including but not limited to, home and industrial refrigerators,deli-meat and cheese counters, refrigerated trucks, and mobilefood-service vehicles.

In a non-limiting embodiment of the invention, the DepositedBacteriophage of the invention is useful in preventing the colonizationof, or inhibiting the growth of, Targeted Bacteria on processed orunprocessed food products by infecting, lysing or inactivating TargetedBacteria present on said food product. Processed or unprocessed foodproducts in which the Deposited Bacteriophage is particularly useful inpreventing the growth or colonization of Targeted Bacteria include, butare not limited to beef (particularly ground beef), food products madewith ground beef such as hamburgers, sloppy joes, lasagna, stews, andother ground beef preparations, fresh vegetables exposed to TargetedBacteria presumably via animal waste, such as lettuce, spinach, greenonions, and other fresh vegetables commonly grown out of doors infields, drinking water, and foodstuffs secondarily contaminated withTargeted Bacteria through contact with contaminated foods, sewage, oranimal feces.

The Deposited Bacteriophage can also be administered to potable andnon-potable water sources to reduce or eliminate the presence ofTargeted Bacteria.

Bacteriophage compositions of the invention may be provided in aqueousor non-aqueous embodiments for the preservation of food.

Aqueous embodiments of the Deposited Bacteriophage include aqueouscompositions comprising, or alternatively consisting of, the DepositedBacteriophage alone or in combination with other bacteriophage orbacteriophages. Aqueous embodiments of the Deposited Bacteriophage areavailable in solutions that include, but are not limited to, phosphatebuffered saline, Luria-Bertani Broth or chlorine-free water.

Non-aqueous embodiments of the Deposited Bacteriophage include, but arenot limited to, lyophilized compositions or spray-dried compositionscomprising, or alternatively consisting of, the Deposited Bacteriophagealone or in combination with other bacteriophage(s). Freeze-dried andspray-dried compositions may also include soluble and/or insolublecarrier materials as, for example, processing aids.

The Deposited Bacteriophage can be administered at a concentrationeffective to prevent the initial colonization of foods with TargetedBacteria, or to inhibit the growth or colonization of food or foodproducts, as well as the equipment used to process or store food. In anon-limiting embodiment of the invention, the Deposited Bacteriophagestypically administered at a growth inhibiting effective amount of aconcentration of about 10⁷ to about 10¹¹ Plaque Forming Units (PFU)/ml.One of skill in the art is capable of ascertaining bacteriophageconcentrations using widely known bacteriophage assay techniques (Adams,M. H. (1959). Methods of study bacterial viruses. Bacteriophages.London, Interscience Publishers, Ltd.: 443-519.). The Depositedbacteriophage at such concentrations may be applied at, for example,about 1 ml/500 cm² of food surface.

Environmental Control

In another embodiment of the invention, the Deposited Bacteriophage isadministered to environments to control the growth or viability ofTargeted Bacteria. Environments in which the Deposited Bacteriophage isuseful to control the growth or viability of Targeted Bacteria include,but are not limited to, abattoirs, meat processing facilities, feedlots,vegetable processing facilities, medical facilities (includinghospitals, out-patient clinics, school and/or university infirmaries,and doctors offices), military facilities, veterinary offices, animalhusbandry facilities, public and private restrooms, and nursing andnursing home facilities. The invention further contemplates the use ofthe Deposited Bacteriophage for the battlefield decontamination of foodstuffs, the environment, and personnel and equipment, both military andnon-military.

The Deposited Bacteriophage is additionally useful alone or incombination with other bacteriophage(s) and/or other compounds, forpreventing the formation of biofilms, or controlling the growth ofbiofilms, in various environments. Aqueous embodiments of the DepositedBacteriophage are available in solutions that include, but are notlimited to, phosphate buffered saline, Luria-Bertani Broth orchlorine-free water. In a particularly preferred embodiment, theDeposited Bacteriophage is used to control biofilm formation and growthin municipal water systems, industrial water systems, and personal watersystems, as well as biofilms present in refrigerated environments.

The modes of administration include, but are not limited to, spraying,hosing, and any other reasonable means of dispersing aqueous ornon-aqueous Bacteriophage compositions, in an amount sufficiently highto inhibit the growth or viability of Targeted Bacteria. In anon-limiting embodiment of the invention, the Deposited Bacteriophage isuseful in preventing the growth or viability of Targeted Bacteria byinfecting, lysing or inactivating Targeted Bacteria present in saidenvironment. Administration of the Deposited Bacteriophage compositionincludes application to the floors, walls, counter-tops, ceilings,drains or any other environmental surface.

Bacteriophage compositions of the invention are available in aqueous ornon-aqueous embodiments discussed earlier for Food Preservationapplications.

In another embodiment of the invention, the Deposited Bacteriophage isadded as a component of paper products, either during processing orafter completion of processing of the paper products. Paper products towhich the Deposited Bacteriophage may be added include, but are notlimited to, paper towels, toilet paper, and moist paper wipes. In apreferred embodiment of the invention, the Deposited Bacteriophage isadded as a component of cleansing wipes; it may be added in an aqueousstate to a liquid-saturated paper product, or alternatively may be addedin powder form such as a lyophilized preparation, to dry paper products,or any combination thereof.

The Deposited Bacteriophage can be administered at a concentrationeffective to inhibit the growth or viability of Targeted Bacteria in aparticular environment. In a non-limiting embodiment of the invention,the Deposited Bacteriophage is administered at a concentration of about10⁷ to 10¹¹ PFU/ml. One of skill in the art is capable of ascertainingbacteriophage concentrations using widely known bacteriophage assaytechniques (Adams, M. H. (1959). Methods of study bacterial viruses.Bacteriophages. London, Interscience Publishers, Ltd.: 443-519.).

Prevention or Treatment of Infection or Colonization

In another embodiment, the invention contemplates a method for theprevention or treatment of illnesses caused by the Targeted Bacteria,comprising contacting a microbial growth inhibiting effective amount ofa bacteriophage composition comprising the Deposited Bacteriophage witha site or sites of infection of a host mammal infected with TargetedBacteria.

The infected mammalian host may be a human host or animal host. Inparticular, the host may be a bovine, poultry, or porcine host.Prevention of the infection by Targeted Bacteria, or treatment ofinfected persons or animals, is particularly preferred inimmuno-compromised persons, pregnant females, and newborns and infants,who maybe at an elevated risk of infection by Targeted Bacteria. Themodes of contact include, but are not limited to, spraying or mistingthe bacteriophage composition on the infected mammalian host, byinjecting at a site or sites of infection a pharmaceutically acceptablecomposition containing a concentration of the Deposited Bacteriophagesufficiently high to inhibit the growth of Targeted Bacteria, or byingesting a solution containing a concentration of the DepositedBacteriophage sufficiently high to inhibit the growth of TargetedBacteria. Additional routes of administration include but are notlimited to oral, rectal, topical, ophthalmic, buccal, intravenous,optic, nasal, vaginal, inhalation, and intrapleural.

In another nonlimiting embodiment of the invention, the DepositedBacteriophage is useful for preparing bacterial vaccines or bacterinsthat eliminate or reduce colonization of the Targeted Bacteria in,and/or their being shed by, various agriculturally-important animals.One example of a practical application for that type of vaccine is inthe cattle-raising industry, where its administration may significantlyreduce colonization of cattle with the Targeted Bacteria; thus,improving public safety by reducing contamination of beef with theTargeted Bacteria.

Bacteriophage compositions of the invention are available in aqueous ornon-aqueous embodiments discussed earlier for Food Preservationapplications.

The Deposited Bacteriophage can be administered at a concentrationeffective to inhibit the growth or viability of Targeted Bacteria in theinfected host. In a non-limiting embodiment of the invention, theDeposited Bacteriophage is administered at a concentration of about 10⁷to 10¹¹ PFU/ml. One of skill in the art is capable of ascertainingbacteriophage concentrations using widely known bacteriophage assaytechniques (Adams, M. H. (1959). Methods of study bacterial viruses.Bacteriophages. London, Interscience Publishers, Ltd.: 443-519.)

Depending on the severity of peculiarities of the infection, theDeposited Bacteriophage can be administered to animals (includinghumans) (i) orally, in tablet or liquid formulation (105-1011 PFU/dose),(ii) rectally, (iii) locally (skin, eye, ear, nasal mucosa, etc.), intampons, rinses and creams, (iv) as aerosols or intrapleunal injectionsand (v) intravenously.

Use of Recombinant Bacteriophage

In one embodiment of the invention, homologous recombination techniquesare used to introduce sequences encoding alternative proteins,non-functional proteins, or non-coding sequences into the bacteriophageDNA sequence. Such techniques are useful to “knock-out” undesired traitsof the Deposited Bacteriophage, or alternatively to introduce differenttraits. In a particularly preferred embodiment of the invention,homologous recombination is used to “knock-out” ORFs encoding proteinsthat maybe involved in a lysogenic cycle of the bacteriophage.

In another embodiment of the invention, homologous recombination is usedto introduce or knock-out genes involved in burst size. For example,homologous recombination is used to introduce alternative bacteriophagegenes which delay the burst event or increase the phage burst size.References disclosing alternative bacteriophage genes involved in thetiming of the burst event or the size of the phage burst include, butare not limited to (Johnson-Boaz, R., C. Y. Chang, et al. (1994). “Adominant mutation in the bacteriophage lambda S gene causes prematurelysis and an absolute defective plating phenotype.” Mol Microbiol 13(3):495-504; Wang, I. N., D. L. Smith, et al. (2000). “Holins: the proteinclocks of bacteriophage infections.” Annu Rev Microbiol 54: 799-825).

In another embodiment of the invention, recombinant bacteriophageharboring reporter system(s) is generated for various practicalapplications. One example of possible application of such system isspecies identification/confirmation of Targeted Bacteria as bacterialdiagnostics. Another possible application is the detection of thepresence of viable cells of Targeted Bacteria to which the Depositedbacteriophage have specificity. Following the techniques of Loessner etal., for example, one of skill in the art can generate recombinantreporter bacteriophage (Loessner, M. J., C. E. Rees, et al. (1996).“Construction of luciferase reporter bacteriophage A511::luxAB for rapidand sensitive detection of viable Listeria cells.” Appl EnvironMicrobiol 62(4): 1133-1140.). For example, the Vibrio harveyi luxAB genemay be introduced into the bacteriophage DNA sequence using techniquessuch as homologous recombination. An ideal target for the introductionof the luxAB gene is immediately downstream and in frame with an ORFencoding bacteriophage capsid protein, thereby creating a sequenceencoding a fusion protein. The preferable location of introduction ofthe luxAB gene sequence is particularly before any sequence encoding atranscriptional terminator downstream of the ORF encoding a capsidprotein. Other bacteriophage ORF sequences which may function as usefulsources of luxAB gene-fusions include gene sequences encodingtail-sheath proteins, or any other late gene region sequences encodingphage head or tail proteins. The resulting recombinant bacteriophage maybe used with methods of the invention to detect the presence of viablecells of Targeted Bacteria.

In addition to the Vibrio harveyi luxAB gene, other reporter genes whichare useful for the generation of reporter bacteriophage include, but arenot limited to, the firefly luciferase gene.

The invention further contemplates the introduction of one or more ofthe above-described recombinant events. For example, a recombinantbacteriophage of the invention may harbor one or more reporter gene(s)as well as lack one or more genes associated with certain undesirablebiological functions of the bacteriophage.

Use of Bacteriophage Derivatives

Derivatives, such as polypeptides, including but not limited tobacteriophage lytic enzymes, encoded by the bacteriophage or thebacteriophage progeny are used for applications designed to prevent thegrowth of Targeted Bacteria through cell wall lysis. In this context,lytic polypeptides are useful for the prevention of the growth ofTargeted Bacteria on processed and unprocessed food products, as well asequipment used for the processing of said food products.

In another preferred embodiment of the invention, bacteriophagederivatives are useful for the treatment of one or more infections in amammal, including humans, by administering their therapeuticallyeffective amounts to the patient. This method is useful for thetreatment of infections of the gastrointestinal system. Similarly, thismethod is useful in a prophylactic setting for the prevention ofinfection by Targeted Bacteria in pregnant mammals, including humans.This method of treatment is further useful for the prevention or otherdisorders or infections caused by Targeted Bacteria, such as acutebloody or non-bloody diarrhea, sometimes associated withhemolytic-uremic syndrome.

Another nonlimiting embodiment of the invention is that thebacteriophage derivatives such as lysins will be useful for preparingbacterial vaccines or bacterins that eliminate or reduce colonization ofthe Targeted Bacteria in, and/or their being shed by, variousagriculturally-important animals. One example of a practical applicationfor that type of vaccine is in the cattle-raising industry, whereadministration of such vaccines/bacterins may significantly reducecolonization of cattle with the Targeted Bacteria; thus, improvingpublic safety by reducing contamination of beef with the TargetedBacteria.

Detection Systems

The Deposited bacteriophage, its progeny, recombinant bacteriophage, orderivatives of the above are useful in methods of screeningenvironmental samples (including food products and food processingequipment) and clinical specimens for the presence of viable cells ofTargeted Bacteria. For example, in one such system, recombinantbacteriophage containing a reporter system such as, for example, aluciferase reporter system is applied to the sample and analyzed at sometime point in the future for the activation of the reporter molecule.The activation of the reporter molecule is indicative of the presence ofviable cells of Targeted Bacteria.

The Deposited bacteriophage, its progeny, recombinant bacteriophage, orderivatives such as lytic enzymes are useful in methods of screeningenvironmental samples including food products and food processingequipment and clinical specimens for the presence of viable cells ofTargeted Bacteria, by monitoring and measuring bacterial metabolismproducts such as bacterial adenosine kinase (AK) or adenosinetriphosphate (ATP) released as a result of specific lysis of TargetedBacteria. For example, when the released ATP is incubated with aluciferin/luciferase mixture, a rapid flash of peak light emissionoccurs within less than a second, followed by a steady glow lasting forseveral hours. By measuring the luminescence, it is possible to obtain aquantitative estimate of the number of bacterial cells in a sample.Although the basic approach involved in such detection-based assays isfairly well-established, the existing assays have shortcomings thathinder their wide acceptance. For example, the various reagents thathave been used to lyse bacteria and release their ATP havebroad-specificity; therefore, ATP is released from all susceptiblebacterial and eukaryotic cells present in the sample, which can causefalse-positive readings. In this context, the original DepositedBacteriophage, its progeny, recombinant bacteriophage, or derivativessuch as lytic enzymes will specifically lyse Targeted Bacteria withoutaffecting any other prokaryotic or eukaryotic cells that may be presentin the sample, thus providing means for accurately and specificallyidentifying and detecting Targeted Bacteria.

Epidemiological Typing

The Deposited Bacteriophage, and/or its progeny and derivatives may befurther useful as a tool for the epidemiological typing of TargetedBacteria. For example, one of skill in the art can use the DepositedBacteriophage of the invention to screen a panel of Targeted Bacteriaisolates to aid in the taxonomic identification of the TargetedBacteria, by determining which isolates yield a positive lytic reactionto the Deposited bacteriophage. For example, see (van der Mee-Marquet,N., M. Loessner, et al. (1997). “Evaluation of seven experimental phagesfor inclusion in the international phage set for the epidemiologicaltyping of Listeria monocytogenes.” Appl Environ Microbiol 63(9):3374-3377.).

Preparation of Vaccines or Bacterins

The Deposited Bacteriophage, and/or its progeny and derivatives, alsomay be valuable for preparing bacterial lysates to be used as vaccinesor bacterins. The immunogenicity of such vaccines or bacterins may besuperior to that of so-called dead cell vaccines because phage-mediatedlysis is a more effective and gentler approach for exposing protectiveantigens of bacteria than are approaches used to prepare the lattervaccines. For example, methods commonly used to inactivate bacterialpathogens for dead-cell vaccines, including but not limited to heattreatment, UV-irradiation, and chemical treatment, may deleteriouslyaffect a vaccine's effectiveness by reducing the antigenicity ofrelevant immunological epitopes (Holt, M. E., M. R. Enright, et al.(1990). “Immunisation of pigs with killed cultures of Streptococcus suistype 2.” Res Vet Sci 48(1): 23-27; Melamed, D., G. Leitner, et al.(1991). “A vaccine against avian colibacillosis based on ultrasonicinactivation of Escherichia coli.” Avian Dis 35(1): 17-22; Lauvau, G.,S. Vijh, et al. (2001). “Priming of memory but not effector CD8 T cellsby a killed bacterial vaccine.” Science 294(5547): 1735-1739). Thepresence of viable bacteriophage may also serve as an additionalefficacy-enhancing factor, increasing the effectiveness of a phagelysate via their antibacterial effect on the Targeted Bacteria.

The above description of various illustrated embodiments of theinvention is not intended to be exhaustive or to limit the invention tothe precise form disclosed. While specific embodiments of, and examplesfor, the invention are described herein for illustrative purposes,various equivalent modifications are possible within the scope of theinvention, as those skilled in the relevant art will recognize. Theinvention may be practiced in ways other than those particularlydescribed in the foregoing description and examples. The teachingsprovided herein of the invention can be applied to other purposes, otherthan the examples described below.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, manuals, books, or otherdisclosures) in the Background of the Invention, Detailed Description,and Examples is herein incorporated by reference in their entireties.

Tables

Table 1 shows the lytic specificity of the bacteriophage for E. coliO157:H7, the Targeted Bacteria.

Table 2 shows the lytic specificity of the bacteriophage fornon-Targeted Bacteria of the same bacterial species.

Tables 2 and 3 shows the lytic specificity of the bacteriophage fornon-Targeted Bacteria of other Gram-positive and Gram-negative bacterialspecies.

TABLE 1 Lytic activity of component monophages against O157:H7 strainsof E. coli Bacterial strains # Intralytix ID Original ID ECML-4Source/Comments 1 Ec121 KSU 1 + University of Maryland 2 Ec122 KSU 2 +University of Maryland 3 Ec123 KSU 3 − University of Maryland 4 Ec124KSU 4 − University of Maryland 5 Ec125 KSU 5 − University of Maryland 6Ec126 KSU 6 + University of Maryland 7 Ec127 KSU 7 − University ofMaryland 8 Ec129 EHEC-1; ATCC 43895 − 1982 outbreak strain 9 Ec13093-111; EHEC-2 + 1993 outbreak strain 10 Ec131 OK-1; EHEC-3 − 1996outbreak strain 11 Ec132 86-24; EHEC-4 + Sporadic case 12 Ec133 2886-75;EHEC-5 + First US case of disease 13 Ec134 493/89; EHEC-6 +Sorbitol-fermenting strain 14 Ec135 E32511; EHEC-7 + Nonmotile strain 15Ec136 G5101; EHEC-8 + Glucuronidase+ strain 16 Ec141 gfb86 + ARS USDA,Beltsville 17 Ec149 87-23 + University of Health Sciences 18 Ec150ATCC700728 + QC strain for Chromagar 19 Ec232 EC00002 + Human isolate;Allegheny County 20 Ec233 EC00003 − Human isolate; Allegheny County 21Ec234 EC00004 − Human isolate; Allegheny County 22 Ec235 EC00005 + Humanisolate; Allegheny County 23 Ec236 EC00007 + Human isolate; AlleghenyCounty 24 Ec237 EC00008 + Human isolate; Allegheny County 25 Ec238EC00009 + Human isolate; Allegheny County 26 Ec239 EC00010 + Humanisolate; Allegheny County 27 Ec240 EC00011 + Human isolate; AlleghenyCounty 28 Ec241 EC00012 + Human isolate; Allegheny County 29 Ec242EC00013 + Human isolate; Allegheny County 30 Ec243 EC00014 + Humanisolate; Allegheny County 31 Ec244 EC00015 + Human isolate; AlleghenyCounty 32 Ec245 EC00016 + Human isolate; Allegheny County 33 Ec246EC00017 + Human isolate; Allegheny County 34 Ec247 EC00018 − Humanisolate; Allegheny County 35 Ec248 EC00019 + Human isolate; AlleghenyCounty 36 Ec249 EC00020 + Human isolate; Allegheny County 37 Ec250EC00021 − Human isolate; Allegheny County 38 Ec251 EC00022 − Humanisolate; Allegheny County 39 Ec252 EC00023 + Human isolate; AlleghenyCounty 40 Ec253 EC00024 + Human isolate; Allegheny County 41 Ec254EC00025 − Human isolate; Allegheny County 42 Ec255 EC00026 + Humanisolate; Allegheny County 43 Ec256 EC00027 + Human isolate; AlleghenyCounty 44 Ec257 EC00028 + Human isolate; Allegheny County 45 Ec258EC00029 − Human isolate; Allegheny County 46 Ec259 EC00030 − Humanisolate; Allegheny County 47 Ec260 EC00031 − Human isolate; AlleghenyCounty 48 Ec261 EC00033 + Human isolate; Allegheny County 49 Ec262EC00034 + Human isolate; Allegheny County 50 Ec263 EC00035 + Humanisolate; Allegheny County 51 Ec264 EC00036 + Human isolate; AlleghenyCounty 52 Ec265 EC00037 + Human isolate; Allegheny County 53 Ec266EC00038 − Human isolate; Allegheny County 54 Ec267 EC00039 − Humanisolate; Allegheny County 55 Ec268 EC00040 + Human isolate; AlleghenyCounty 56 Ec269 EC00041 − Human isolate; Allegheny County 57 Ec270EC00042 − Human isolate; Allegheny County 58 Ec271 EC00043 − Humanisolate; Allegheny County 59 Ec272 EC00044 − Human isolate; AlleghenyCounty 60 Ec273 EC00045 + Human isolate; Allegheny County 61 Ec274EC00046 − Human isolate; Allegheny County 62 Ec275 EC00047 + Humanisolate; Allegheny County 63 Ec276 EC00048 + Human isolate; AlleghenyCounty 64 Ec277 EC00049 − Human isolate; Allegheny County 65 Ec278EC00050 − Human isolate; Allegheny County 66 Ec279 EC00051 + Humanisolate; Allegheny County 67 Ec280 EC00052 + Human isolate; AlleghenyCounty 68 Ec281 EC00053 + Human isolate; Allegheny County 69 Ec282EC00054 + Human isolate; Allegheny County 70 Ec283 EC00055 + Humanisolate; Allegheny County 71 Ec284 EC00056 + Human isolate; AlleghenyCounty 72 Ec285 EC00057 + Human isolate; Allegheny County 73 Ec286EC00058 + Human isolate; Allegheny County 74 Ec287 EC00059 + Humanisolate; Allegheny County 75 Ec288 EC00060 + Human isolate; AlleghenyCounty 76 Ec289 EC00061 + Human isolate; Allegheny County 77 Ec290EC00062 + Human isolate; Allegheny County 78 Ec291 EC00063 + Humanisolate; Allegheny County 79 Ec292 EC00064 + Human isolate; AlleghenyCounty 80 Ec293 EC00065 + Human isolate; Allegheny County 81 Ec294EC00066 + Human isolate; Allegheny County 82 Ec295 EC00067 + Humanisolate; Allegheny County 83 Ec296 EC00068 + Human isolate; AlleghenyCounty 84 Ec297 EC00069 − Human isolate; Allegheny County 85 Ec298EC00070 + Human isolate; Allegheny County 86 Ec299 EC00071 + Humanisolate; Allegheny County 87 Ec300 EC00072 + Human isolate; AlleghenyCounty 88 Ec301 EC00073 + Human isolate; Allegheny County 89 Ec302EC00074 − Human isolate; Allegheny County 90 Ec303 EC00075 + Humanisolate; Allegheny County 91 Ec304 EC00076 + Human isolate; AlleghenyCounty 92 Ec305 EC00077 − Human isolate; Allegheny County 93 Ec306EC00078 − Human isolate; Allegheny County 94 Ec307 EC00079 + Humanisolate; Allegheny County 95 Ec308 EC00080 + Human isolate; AlleghenyCounty 96 Ec309 EC00081 − Human isolate; Allegheny County 97 Ec310EC00082 + Human isolate; Allegheny County 98 Ec311 EC00083 − Humanisolate; Allegheny County 99 Ec312 EC00376 + Human isolate; AlleghenyCounty 100 Ec313 EC00377 − Human isolate; Allegheny County 101 Ec314EC00378 + Human isolate; Allegheny County 102 Ec315 EC00379 − Humanisolate; Allegheny County 103 Ec316 EC00380 − Human isolate; AlleghenyCounty 104 Ec317 EC00382 + Human isolate; Allegheny County 105 Ec318EC00383 + Human isolate; Allegheny County 106 Ec319 EC00384 + Humanisolate; Allegheny County 107 Ec320 EC00385 + Human isolate; AlleghenyCounty 108 Ec321 EC00398 + Human isolate; Allegheny County 109 Ec322EC00499 + Human isolate; Allegheny County 110 Ec323 EC00500 + Humanisolate; Allegheny County 111 Ec324 EC00501 + Human isolate; AlleghenyCounty Total lysed: number of strains (percent) 78 (70%) + Lysed byphage − Not lysed by phage

TABLE 2 Lytic activity of ECML-4 phage against non-O157:H7 strains of E.coli Bacterial strains Phage # Intralytix ID Original ID ECML-4 − 1Ec156 ECOR-01; ATCC 35321 − 2 Ec157 ECOR-02; ATCC 35322 − 3 Ec158ECOR-03; W1R1(a) − 4 Ec159 ECOR-04; RM39A − 5 Ec160 ECOR-05; RM60A − 6Ec161 ECOR-06; RM66C − 7 Ec162 ECOR-07; RM73C − 8 Ec163 ECOR-08; RM77C −9 Ec164 ECOR-09; FN98 − 10 Ec165 ECOR-10; ANI − 11 Ec166 ECOR-11; C97 +12 Ec167 ECOR-12; FN59 − 13 Ec168 ECOR-13; FN10 − 14 Ec169 ECOR-14; P62− 15 Ec170 ECOR-15; FN3 − 16 Ec171 ECOR-16; ATCC 35335 − 17 Ec172ECOR-17; RM200Q − 18 Ec173 ECOR-18; RM210F − 19 Ec174 ECOR-19; RM21OJ −20 Ec175 ECOR-20; RM213J − 21 Ec176 ECOR-21; RM213K − 22 Ec177 ECOR-22;RM215C − 23 Ec178 ECOR-23; ATCC 35342 − 24 Ec179 ECOR-24; ATCC 35343 −25 Ec180 ECOR-25 − 26 Ec181 ECOR-26; LL − 27 Ec182 ECOR-27; RM24J − 28Ec183 ECOR-28; RM52B − 29 Ec184 ECOR-29; RM3A − 30 Ec185 ECOR-30; RM10A− 31 Ec186 ECOR-31; RM12 − 32 Ec187 ECOR-32; ATCC 35351 − 33 Ec188ECOR-33; ATCC 35352 − 34 Ec189 ECOR-34; WIR2(a) − 35 Ec190 ECOR-35;RM42B − 36 Ec191 ECOR-36; RM77B − 37 Ec192 ECOR-37; RM44B − 38 Ec193ECOR-38; RM75A − 39 Ec194 ECOR-39; FN104 − 40 Ec195 ECOR-40; P60 − 41Ec196 ECOR-41; T44 − 42 Ec197 ECOR-42; DAR1 − 43 Ec198 ECOR-43; FN36 −44 Ec199 ECOR-44; RM189I − 45 Ec200 ECOR-45; RM201C − 46 Ec201 ECOR-46;RM202F − 47 Ec202 ECOR-47; RM211C − 48 Ec203 ECOR-48; C90 − 49 Ec204ECOR-49; FN90 − 50 Ec205 ECOR-50; P97 − 51 Ec206 ECOR-51; DD − 52 Ec207ECOR-52; RM73A − 53 Ec208 ECOR-53; RM33B − 54 Ec209 ECOR-54; RM64A − 55Ec210 ECOR-55; FN4 − 56 Ec211 ECOR-56; ATCC 35375 − 57 Ec212 ECOR-57;ATCC 35376 − 58 Ec213 ECOR-58; RM185S − 59 Ec214 ECOR-59; SIL8 − 60Ec215 ECOR-60; C89 − 61 Ec216 ECOR-61; FN83 − 62 Ec217 ECOR-62; P69 − 63Ec218 ECOR-63; FN21 − 64 Ec219 ECOR-64; C70 − 65 Ec220 ECOR-65; RM202I −66 Ec221 ECOR-66; ATCC 35385 − 67 Ec222 ECOR-67; RM217T − 68 Ec223ECOR-68; RM224H − 69 Ec224 ECOR-69; RM45EM − 70 Ec225 ECOR-70; ATCC35389 − 71 Ec226 ECOR-71; ABU84 − 72 Ec227 ECOR-72; P68 − 73 Ec137EHEC-9; 5905 − 74 Ec138 TB182A; EHEC-10 − 75 Ec139 DEC5D; EHEC-11 − 76Ec140 3256-97; EHEC-12 − Total lysed: number of strains (percent) 1(1.3%) + Lysed by phage − Not lysed by phage

TABLE 3 ECML-4 phage did not lyse S. aureus, L. monocytogenes,Salmonella, and P. aeruginosa Bacterial strains Phage Intralytix IDOriginal ID Species ECML-4 − 1 Lm253 ATCC 35152 L. monocytogenes − 2Lm254 ATCC 13932 L. monocytogenes − 3 Lm314 ATCC 19117 L. monocytogenes− 4 Lm315 ATCC 19118 L. monocytogenes − 5 Lm317 ATCC 19116 L.monocytogenes − 6 SE566 ATCC 13076 Salmonella Enteritidis − 7 ST567 ATCC13311 Salmonella Typhimurium − 8 SN659 ATCC 6962 Salmonella Newport − 9SS661 ATCC 10719 Salmonella Paratyphi B − 10 SD663 ATCC 15480 SalmonellaDublin − 11 Sa295 9090 S. aureus − 12 Sa296 11287 S. aureus − 13 Sa297ME19 S. aureus − 14 Sa298 ATCC 49775 S. aureus − 15 Sa299 ATCC 14458 S.aureus − 16 Pa27 193 P. aeruginosa − 17 Pa28 190 P. aeruginosa − 18 Pa42 32 P. aeruginosa − 19 Pa71 ATCC 27853 P. aeruginosa − 20 Pa76 ATCC10145 P. aeruginosa − + Lysed by phage − Not lysed by phage

EXAMPLES Example 1 ECML-4 Bacteriophage Isolation

The ECML-4 bacteriophage was isolated from Baltimore Inner Harbor watersusing lysis of the Targeted Bacteria to form plaques in bacterial lawnsas a means of detecting the presence of bacteriophage having lyticspecificity for the Targeted Bacteria. Plaques were harvested, diluted,and re-plated on bacterial lawns through a process of serial enrichmentuntil a single bacteriophage species, or monophage, was obtained asdetermined by a stable restriction fragment length profile of thebacteriophage DNA. The isolates obtained using the technique recitedsupra may be cultured using the techniques as set forth herein. Thebacteriophage was deposited with the ATCC.

Example 2 Deposited Bacteriophage Concentration

Concentration of the Deposited bacteriophage may be determined usingtechniques known in the art (Adams, M. H. (1959). Methods of studybacterial viruses. Bacteriophages. London, Interscience Publishers,Ltd.: 443-519.). When a single phage particle encounters a permissivebacterium it will lyse it with the concomitant release of newly formedphage particles. When phages are mixed with host cells and poured in alayer of soft agar on the surface of a nutrient agar plate supportingbacterial growth, the cells will resume growth. In areas where no phagesare present the bacteria will grow to stationary phase, forming a smoothopaque layer or lawn in the overlay. In areas where phages are present,phage progeny from each infected bacterium will infect neighboringbacteria, resulting in a growing zone of lysis full of liberated phagewhich eventually becomes visible to the naked eye as a plaque in theotherwise smooth bacterial lawn. These plaques can be counted, and theirnumber is widely used for expressing phage titer in plaque-forming unitsor PFU. Using this approach, concentration of the Depositedbacteriophage may be determined. Briefly: (1) Various dilutions of theDeposited bacteriophage preparation are prepared; for example, by mixing0.1 ml of phage sample with 9.9 ml of sterile LB broth. The samples aregently but thoroughly mixed. 0.5 ml of this mixture (which is a 10⁻²dilution of the original sample) is mixed with 4.5 ml of sterile LBbroth (10⁻³ dilution). Several 10-fold dilutions are prepared in asimilar fashion; (2) the contents of the tubes (1 ml of variousdilutions) are transferred into sterile 10 ml culture tubes and 0.1 mlof host bacterial culture are added. The sample is mixed gently beforeproceeding immediately to the next step; (3) 3-5 ml of warm (45-50° C.)0.7% LB agar (top agar) are added. The sample is mixed quickly and verygently. Then, the entire contents of the culture tube are poured onto aplate containing solidified LB agar (bottom agar). The plates are slidin circles a few times on the bench top immediately after pouring; (4)after sitting at room temperature for 10 min to allow the top agar toharden, the plates are inverted and placed into a 37° C. incubator andincubated overnight; (5) the next morning, the number of plaques on theplate with 30-300 individual well spaced plaques are counted and thetiter calculated and expressed as PFU/ml of the starting sample.

Example 3 Production of the Deposited Bacteriophage

The Deposited bacteriophage is produced using a culture system. Morespecifically, strain of the host Targeted Bacteria or otherclosely-related bacterial species on which the bacteriophage canpropagate is cultured in batch culture, followed by inoculation of thebacteriophage at the pre-determined multiplicity of infection (MOI).Following incubation and bacterial lysis, the bacteriophage is harvestedand purified and/or concentrated to yield phage progeny suitable for theuses enumerated herein. Purification and concentration proceduresincluded variously processing through filtration system(s),centrifugation (including continuous-flow centrifugation) or other wellknown bacteriophage purification and concentration techniques (Adams, M.H. (1959). Methods of study bacterial viruses. Bacteriophages. London,Interscience Publishers, Ltd.: 443-519.).

The invention provides compositions comprising active viral particles ofthe bacteriophage capable of lysing strains of Targeted Bacteria. Theconcentration of bacteriophage is determined using phage titrationprotocols. The final concentration of the bacteriophage is adjusted byconcentration, if a more concentrated phage composition is desired, viafiltration, centrifugation, or other means, or by dilution, if a lessconcentrated phage composition is desired, with water or buffer to yielda phage titer of 10⁶ to 10¹² PFU/ml, preferably 10¹⁰ to 10¹¹ PFU/ml. Theresulting bacteriophage compositions are generally stored at 4° C.;alternatively, preparations can be freeze or spray-dried for storage, orcan be encapsulated and stabilized with protein, lipid, polysaccharide,or mixtures thereof. Upon reconstitution, the phage titer can beverified using phage titration protocols and host bacteria. One of skillin the art is capable of determining bacteriophage titers using widelyknown bacteriophage assay techniques (Adams, M. H. (1959). Methods ofstudy bacterial viruses. Bacteriophages. London, IntersciencePublishers, Ltd.: 443-519.).

Example 4 Application of the Deposited Bacteriophage for Preservation ofFood Products

The bacteriophage produced using the methods of the present inventionmay be dispersed in an appropriate aqueous solution or lyophilized orfreeze-dried powder and applied to the surface of food products.Alternatively, the bacteriophage may be included with a cheese cultureor other microbially active foodstuff prior to or during processing.

Example 5 Isolation of the Bacteriophage DNA

Bacteriophage DNA, a derivative of the bacteriophage, can be used forvarious applications such as for preparing DNA-based vaccines, and alsofor analytical purposes, for identifying the bacteriophage such as RFLPprofile determination and comparison. Phage DNA can be isolated using asuitable commercial kit such as the Lambda Mini Kit (Qiagen, Inc.;Valencia, Calif.) or the standard phenol extraction technique. Forexample, 0.75 ml of phage in phosphate-buffered saline solution at atiter of 10⁸-10¹¹, PFU/ml is collected. 10 μl of Proteinase K (20 mg/ml)and 2 μl of RNAse (10 mg/ml) is added, followed by incubation at 37° C.for 30 minutes, and a subsequent incubation at 56° C. for 30 minutes.Following incubation, 75 μl of a mixture of 10% SDS (0.1 ml), 0.5 M EDTA(0.1 ml) and 0.8 ml of water is added and incubated at room temperaturefor 5 min. 0.75 ml of a phenol:chloroform:isoamylalcohol (25:24:1)solution is mixed well with the sample, followed by centrifugation at13,000 RPM for five (5) min. Next, the supernatant (approximately 600μl) is carefully removed and transferred to a clean eppendorf tube. 0.6ml of chloroform is added to the supernatant, mixed well, andcentrifuged at 13,000 RPM for five (5) min. The supernatant is thencarefully extracted (approximately 500 μl). Next, 0.1 volumes of 3Msodium acetate (40 ml) is added to the solution, followed by 2.5 volumesof cold 95% ethanol (1 ml) to precipitate the bacteriophage DNA. Thesolution is allowed to incubate at −20° C. for 1 hour, followed bycentrifugation at 13,000 RPM for thirty (30) min. Followingcentrifugation, the pellet is washed with 1 ml of 70% cold ethanol, andthe supernatant is poured from the pellet. The pellet is allowed to airdry, and is then resuspended in 30-300 μl of TE (10 mM tris-HCL,pH=8.0-8.5, 1 mM EDTA).

Example 6 Restriction Fragment Length Polymorphism (RFLP) Profile

RFLP can be used to identify the Deposited bacteriophage or its progeny.The progeny will have a substantially equivalent RFLP DNA profile as theRFLP DNA profile of the original bacteriophage. A reference RFLP profileof the Deposited bacteriophage is shown in FIG. 1. DNA was isolated fromthe bacteriophage using Qiagen Plasmid Miniprep or Midiprep kits(Valencia, Calif.) according to the manufacturer's directions. The DNAwas quantitated by measuring absorbance at 260 nm. Approximately 0.5-1μg of DNA was digested with an appropriate restriction enzyme (FIG. 1),and RFLP profile was determined on 1% agarose gel after staining withethidium bromide.

Example 7 Lytic Specificity of the Bacteriophage

One hundred eleven E. coli O157:H7 strains were screened for theirsusceptibility to the bacteriophage by the drop-on-lawn method, alsoknown as the “spot test” method. Strains were streaked onto LB agarplates and incubated at 37° C. overnight. One colony of each strain wasinoculated into a separate well of a 96-well microtiter plate containingLB broth and incubated at 37° C. until the OD600 reached 0.2-0.3. Onehundred microliters of each strain were mixed with LB soft agar andpoured onto an LB agar plate. After the soft agar hardened 10 μl of thebacteriophage were spotted in triplicate onto the plates inoculated withthe strains of Targeted Bacteria. Lytic activity was observed afterovernight incubation at 37° C. Lytic specificity results are presentedin Table 1. The bacteriophage lysed 78 (70%) of the 111 strains ofTargeted Bacteria examined. In contrast, it only lysed 1 (1.3%) of 76non-O157:H7 E. coli strains (Table 2) and none of the 20 strains ofother bacterial species (Table 3) examined.

Example 8 Detection of Targeted Bacteria in Food Samples

The bacteriophage or its derivative, such as lytic enzyme, producedusing the methods of the present invention is used to specifically lyseTargeted Bacteria without affecting any other prokaryotic or eukaryoticcells that may be present in the sample; thus, specifically elicitingtheir release of measurable bacterial products such as AK or ATP.Briefly: (1) Samples of the food to be analyzed are obtained andsuspended in appropriate buffer, (2) The Deposited bacteriophage isadded to the suspensions, as a result of which the Targeted Bacteriacells present in the samples are lysed and their ATP is released, (3) Aluciferin+luciferase preparation is added to the mixtures, and (5) Themixtures' luminescence is measured within 60 sec, and the results aredisplayed on a handheld luminometer. It may be possible to establish acorrelation between the luminometer readings and the number of TargetedBacteria cells lysed (in general, the average amount of ATP perbacterial cell is 0.5-1.0 fg; precise correlation between theluminometer readings and the number of Targeted Bacteria cells should beexperimentally established). If Targeted Bacteria cells are not presentin the food samples analyzed, bacterial lysis and ATP-release will notoccur.

Example 9 Preparing Vaccines and Bacterins

One example of utilizing bacteriophages to prepare vaccines andbacterins is to use the lytic Deposited bacteriophage to lyse specificstrains of the Targeted Bacteria, which will yield bacterial lysatescontaining minimally-affected immunological epitopes of the bacteria.The phage may be removed from the final vaccine/bacterin preparation.Alternatively, it may be retained unaltered in the preparation becauseits lytic activity against Targeted Bacteria that may be present in themammalian organism being vaccinated may increase the preparation'sefficacy. In one preferred embodiment of the present invention: (i) themost prevalent, problematic strains of the Targeted Bacteria are chosenso that the vaccine/bacterin contains the immunological epitopes thatare most relevant for protecting against the infection, and (ii) thebacteriophage is kept unaltered in the final vaccine/bacterin, at levelsranging from 10⁶-10¹⁰ PFU/ml.

Bacteriophage-based vaccines and bacterins also may be prepared by usingderivatives of the Deposited bacteriophage to lyse the TargetedBacteria. An example of the general methodology can be briefly outlinedfrom a recent study (Panthel, K., W. Jechlinger, et al. (2003).“Helicobacter pylori ghosts by PhiX protein E-mediated inactivation andtheir evaluation as vaccine candidates.” Infect Immun 71(1): 109-16.) ofan Helicobacter pylori bacterin. The authors used E. coli-H. pylorishuttle plasmid pHel2 and lysis gene e of bacteriophage (pX174 toconstruct H. pylori lysis plasmid pHPC38, which they introduced into H.pylori strain P79. At a pre-determined time, the authors triggered egene-expression in order to elicit bacterial lysis, and they found thatthe lysate protected BALB/c mice against H. pylori infection.

1. An isolated bacteriophage substantially equivalent to ECML-4deposited under ATCC accession No. PTA-7948, said bacteriophage havinglytic activity against E. coli O157:H7 strains.
 2. Isolated progeny ofthe bacteriophage of claim 1, which have RFLP DNA profiles substantiallyequivalent to the profile of said bacteriophage.
 3. A compositioncomprising the isolated bacteriophage of claim
 1. 4. A compositioncomprising the isolated bacteriophage of claim
 2. 5. At least onederivative of an isolated bacteriophage substantially equivalent toECML-4 deposited under ATCC accession No. PTA-7948, said bacteriophagehaving lytic activity against E. coli O157:H7 strains wherein saidderivative comprises nucleic acids, partial or complete genes, geneexpression products, structural components, or one or more combinationsthereof.
 6. At least one derivative of an isolated bacteriophagesubstantially equivalent to ECML-4 deposited under ATCC accession No.PTA-7948. said bacteriophage having lytic activity against E. coliO157:H7 strains wherein said derivative comprises nucleic acids, partialor complete genes, gene expression products, structural components, orone or more combinations thereof.